The broad goals of our research are: to determine the mechanisms by which (Ca2+)i is increased in cardiac myocytes during ATP depletion; to determine if modulation of the extent of Ca2+ loading alters the degree of myocyte injury during ATP depletion; and to determine the causes of persistent alterations of cation homeostasis, contraction, and electrophysiologic function in myocytes after recovery from severe ATP depletion. These studies will be done in isolated cultured neonatal and adult rabbit ventricular myocytes, and in immature and adult human ventricular myocytes obtained by enzymatic dissociation. ATP depletion will be produced by exposure to zero glucose plus metabolic inhibition with cyanide or with cyanide and 2-deoxyglucose. [Ca2+]i ,[Na+]i, and PHi will be measured using fluorescence dyes. Na+ /Ca2+ exchange currents will be quantitated by voltage clamp measurement of currents produced by abrupt exposure of Na+ loaded myocytes to extracellular Ca2+ , and Na+ pump function will be measured by currents produced after abrupt activation of the Na+ /Ka ATPase by resupplying extracellular potassium. Na+ /K+ ATPase ouabain binding will be investigated using 3H-ouabain, and K uptake and K content will be quantified by use of 42k. Membrane potentials will be measured by conventional whole cell patch techniques, and contraction by means of a video motion detector system. With these approaches, we will determine if the increase in [Ca2+]i observed occur during metabolic inhibition in cultured adult rabbit myocytes is inhibited by the exchanger inhibitory peptide, XIP. We will also examine whether stimulation of Na+ /H+ exchange by exposure to angiotensin II or endothelin-1 during metabolic inhibition of adult myocytes with cyanide enhances Na+i- dependent Ca2+ loading, and exacerbates cell injury. Possible developmental- and species- dependent changes in the responses of myocytes to AII and ET-1 will also be investigated. We will determine if Na+ influx mediated by Na-K-Cl co-transport contributes to Na+ loading and enhances injury of adult myocytes during metabolic inhibition, and whether activation of Na- K- Cl co-transport contributes to K+ loss and membrane depolarization during recovery from ATP depletion. We will test the hypothesis that severe ATP depletion produced in cultured adult ventricular myocytes causes an acute and/or persistent reduction in Na+ pump density, and Na+ pump function. We will also determine whether exposure to growth factors present in fetal calf serum will enhance Na+, K+ -ATPase expression and improve function in adult myocytes recovering from an episode of severe ATP depletion. It is hoped that these studies in isolated myocytes will elucidate the pathophysiology and pharmacology of ischemic and reperfusion injury, including the role of Ca2+ in the injury process and the causes of ATP depletion-induced persistent abnormalities in cation homeostasis.
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